Conjugates of antioxidants with metal chelating ligands for use in diagnostic and therapeutic applications

ABSTRACT

The invention provides radiopharmaceuticals for diagnostic and therapeutic applications, conjugates of antioxidants with metal chelating ligands, intermediate compounds, methods of making such radiopharmaceuticals, ligands, and intermediate compounds, and kits for preparing the radiopharmaceutical complexes.

FIELD OF INVENTION

The present invention relates to diagnostic and therapeuticcompositions, methods of their use, and processes of their preparation.

BACKGROUND OF INVENTION

Ascorbic acid (vitamin C) and other antioxidants, such as α-tocopherol,minimize tissue damage caused by oxidative metabolic processes and alsohave acceptable biological tolerance. Recently, an ascorbic acidderivative with antioxidant properties, 2-O-octadecylascorbic acid, hasbeen prepared and has been shown to markedly inhibit the myocardiallesions induced by ischemia-reperfusion treatment in rats. Ascorbic acidmay also bind to the human serum albumin (HSA) weakly with a bindingconstant of about 3.5×10⁴ M⁻¹. Ascorbic acid and other antioxidants havebeen used to stabilize radiopharmaceuticals by decreasing the oxidationof substituents due to radical reactions induced by the decay of theradionuclide.

Metal chelating ligands are designed for use in Nuclear Medicine,Magnetic Resonance Imaging (MRI), and neutron capture therapyapplications. Magnetic resonance (hereinafter sometimes referred to asMR) imaging is widely used for obtaining spatial images of parts of apatient for clinical diagnosis. Typically, the image is obtained byplacing the patient in a strong external magnetic field and observingthe effect of this field on the magnetic properties of protons containedin and surrounding the organ or tissue of the patient. The protonrelaxation times, called T₁ or spin-lattice or longitudinal relaxationtime, and T₂ or spin-spin or transverse relaxation time depend on thechemical and physical environment of the organ or tissue being imaged.In order to improve the clarity of the image, a diagnostic agent isadministered intravenously (hereinafter sometimes referred to as I.V.)and is taken up by the organs, such as the liver, spleen, and lymphnodes to enhance the contrast between healthy and diseased tissues.

The contrast agents used in MR imaging derive their signal-enhancingeffect from the inclusion of a material exhibiting paramagnetic,ferrimagnetic, ferromagnetic or superparamagnetic behavior. Thesematerials affect the characteristic relaxation times of the imagingnuclei in the body regions into which they distribute causing anincrease or decrease in MR signal intensity. There is a need forcontrast agents such as those of the present invention, that selectivelyenhance signal intensity in particular tissue types, as most MR contrastagents are relatively non-specific in their distribution.

Nuclear medicine procedures and treatments are based on internallydistributed radioactive materials, such as radiopharmaccuticals orradionuclides, which emit electromagnetic radiations as alpha or betaparticles or as gamma rays or photons. Following I.V., oral orinhalation administration, ganuna rays are readily detected andquantified within the body using instrumentation such as scintillationand gamma cameras. Compounds derivatized with alpha or beta emitters maybe used for radiotherapeutic applications, providing an internal dose ofcytotoxic radiation at their target tissues(s).

SUMMARY OF THE INVENTION

The invention relates to conjugates of an antioxidant and one or moremetal chelating ligands that may be chelated to radioactive ornon-radioactive metals and use of such conjugates chelated to suchmetals as, for example:

-   -   a) Magnetic resonance diagnostic compositions for visualization        of tissues and compartments that bind or utilize an antioxidant        conjugated to metal chelates;    -   b) Radiodiagnostic compositions for visualization of tissues,        comprising ligands chelated to radioactive gamma-emitting metals        and coupled to said antioxidant conjugates; and    -   c) Compositions for radiotherapy or for neutron capture therapy,        comprising ligands chelated to radioactive alpha or        beta-emitting metals or to metals suitable for neutron capture        therapy and coupled to said antioxidant conjugates.

In one embodiment, the invention provides novel conjugates ofantioxidants and metal chelating ligands. The invention also providesnovel intermediates, methods of making the conjugates and intermediates,methods of stabilizing radiopharmaceutical ligands, and kits forpreparing radiopharmaceuticals. Antioxidants which may be used in thepresent invention include ascorbic acid, para-aminobenzoic acid (PABA),cysteine, monothioglycerol, and gentisic acid. Ascorbic acid is apreferred antioxidant of the invention.

In a preferred embodiment, the invention provides a compound having thefollowing chemical structure:

wherein M is ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁶⁸yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y,⁸⁶Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb,²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ²¹⁵Bi, ¹⁷⁷Lu, chromium (III), manganese(II), iron (II), iron (III), cobalt (II), nickel (II), copper (II),praseodymium (III), neodymium (III), samarium (III), gadolinium (III),terbium, (III), dysprosium (III), holmium (III), erbium (III) orytterbium (III); and X is CH₂, an amino acid, a peptide, a protein, oran antibody.

In one preferred embodiment, X is CH₂. In another preferred embodiment,X is the amino acid represented by the chemical structure:

In certain preferred embodiments, the metal (M) is ^(99m)Tc, ¹⁸⁶Re,¹⁸⁸Re, ⁹⁰Y, ⁸⁸Y, ⁸⁶Y, ¹⁷⁷Lu, or gadolinium (III).

In another embodiment, the invention provides kits for preparing aradiopharmaceutical. Kits of the invention include an oxidant covalentlybound to a complexing (or radiopharmaceutical ligand). In preferredembodiments, the kit includes a targeting molecule bound to theantioxidant, the ligand, or, most preferably, both. In certain of theseembodiments, the targeting molecule is an amino acid, a peptide, aprotein, or an antibody.

In yet another embodiment, the invention provides methods of stabilizinga radiopharmaceutical ligand, which optionally includes a targetingmolecule, by conjugating the radiopharmaceutical ligand with anantioxidant.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used in this specification: “DOTA” means1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; “HATU” meansO-(7-azabelzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; “DIEA” means diisopropylethylamine; “DMF” meansN,N-dimethylformamide; “TsCl” means p-toluenesulfonyl chloride; “THF”means tetrahydrofuran; “TFA” means trifluoroacetic acid; and “RT” meansroom temperature. In addition, the terms “chelating ligand,” “complexingligand,” and “radiopharmaceutical ligand” are used interchangeablythroughout this specification, except where the context requiresotherwise.

The present invention is directed, in part, to conjugates of anantioxidant, such as ascorbic acid, and one or more polydentatemacrocyclic or non-macrocyclic metal-chelating ligand residues that areoptionally chelated to radioactive or non-radioactive metals capable ofeither being detected by imaging means for diagnosis or capable ofproviding a therapeutic or radiotherapeutic effect. The metal chelatinggroups can be either macrocyclic or non-macrocyclic multidentate metalchelating ligands, and the structure of these ligands and the metalsthat are chelated to them may be varied depending on the use envisionedfor them. For example, for compounds of the present application that areused for Magnetic Resonance Imaging applications, chelating polyazamacrocyclic ligands that form stable compounds with superparamagnetic orparamagnetic metals, and chelating ligands that provide enhancedrelaxivity properties (vide infra) are preferred. For such applications,gadolinium is the preferred metal.

In a further embodiment, conjugates of the present invention may be usedfor radiodiagnostic or radiotherapeutic purposes. In this application,an antioxidant, such as ascorbic acid, is conjugated to a chelatingligand, which form stable complexes with radioactive metals. Thechelating ligands that may be used in the practice of the presentinvention are not particularly limited and are well known to thoseskilled in the art. Such ligands include, for example, Oxa-PnAO ligandsand peptide analogue chelators, such as those with an N₃S configuration.Radioactive metals include the elements having atomic numbers of 22 to29, 42, 44 and 58-70. For example, radioactive isotopes include:^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁶⁸Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ⁸⁶Y, ¹⁵³Sm,¹⁶⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷RU, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi,²¹²Bi, ²¹³Bi, ²¹⁴Bi, ²¹⁵Bi, and ¹⁷⁷Lu. The choice of metal ion will bedetermined based on the desired therapeutic or diagnostic application.Where ^(99m)Tc is the radioactive metal used, Oxa-PnAO ligands orN,N-Me₂-Gly-Ser-Cys-Gly are preferably used to form conjugates with anantioxidant, such as ascorbic acid.

The antioxidants used in the invention are not particularly limited,provided that the antioxidant can be conjugated to a ligand and/ortargeting molecule, as described herein. For example, antioxidants whichmay be used in the present invention include ascorbic acid,para-aminobenzoic acid (PABA), cysteine, monothioglycerol, and gentisicacid. Of these, ascorbic acid is preferred.

The conjugates may further comprise targeting molecules such as, forexample, proteins, peptides and antibodies that localize to desiredareas of the body. Preferred targeting molecules are peptides oranalogues thereof and may include a monomer or multimer of one or morepeptides. Examples of suitable targeting molecules include gastrinreleasing peptide (GRP) agonists, such as those disclosed in U.S. Pat.No. 6,200,546, incorporated herein by reference in its entirety. Otherusefuil targeting molecules include those disclosed in U.S. Pat. Nos.5,662,885; 5,780,006; and 5,976,495, incorporated herein by reference intheir entirety, and particularly monomers or multimers of TKPPR oranalogues thereof Analogues of a peptide include molecules that targetthe peptide's receptor with the same or greater avidity, as well asmuteins, retropeptides and retroinversion peptides. One of ordinaryskill will appreciate that these analogues may also containmodifications such as substitutions, deletions and/or additions of oneor several amino acids, insofar as these modifications do not alter thebiological activity of the peptide in a significantly negative manner.

General structures of Oxa-PnAO ligands are detailed in U.S. Pat. No.6,093,382, which is incorporated by reference herein. These conjugatesare intended for preparation of compounds for use in nuclear medicineand radiotherapy applications and are based on the general oxa-PnAOligand class described in U.S. Pat. No. 5,608,110, which areincorporated by reference herein. For diagnostic applications, ^(99m)Tcis the preferred metal.

Structures and preparation of peptide-derived N₃S radionuclide chelatorsare discussed in U.S. Pat. Nos. 5,662,885; 5,780,006 and 5,976,495, eachof which is incorporated herein by reference in its entirety.Particularly preferred N₃S chelators are N,N-dimethyl-Gly-Ser-Cys-Glyand N,N-dimethyl-Gly-t-butylGly-Cys-Gly.

Radiopharmaceutical conjugates (either diagnostic or therapeutic) of thepresent invention confer the added benefit of introducing an antioxidant(such as ascorbic acid) in close proximity to the oxidizable groups onthe radiopharmaceutical (either diagnostic or therapeutic). Suchoxidizable groups may include, for example, peptides containingmethionine or free thiols. Further, such oxidizable groups may belocated on either the ligand or a targeting molecule. This covalentattachment of an antioxidant and the chelator (optionally coupled to atargeting ligand) provides additional stability due to the closeproximity of the antioxidant to substituents on the radiopharmaceuticalthat are susceptible to oxidation induced by the decay of theradionuclide. Indeed, it has been reported that the ester of 6-hydroxyascorbic acid retains a number of useful antioxidant properties. Theamide bond introduced into 6-hydroxy ascorbic acid in the compoundsdisclosed herein is expected to have greater serum stability than theester compounds previously disclosed, and thus, to exhibit antioxidantbehavior. Therefore, the ascorbic acid or other antioxidant derivativesof the invention are expected to retain their antioxidant propertieswhen conjugated to the chelator and/or targeting molecules, therebyimproving the stability of the conjugates.

Specifically, where a targeting ligand is used, the antioxidant may beattached to the targeting molecule, which is attached to the chelatingligand. For example, where ascorbic acid is the antioxidant used,6-amino ascorbic acid may be attached to the C-terminus of a peptidetargeting molecule or via the beta or gamma carboxyl group of anaspartic or glutamic acid in the peptide. Similarly, the 6-aminoascorbic acid could be attached to the N-terminus of the peptide via adi-carboxylic acid such as succinic acid.

Alternatively, in the absence of a targeting molecule, the antioxidantmay be attached to the chelating ligand as shown herein, or usingmethods known to those skilled in the art.

Examples of paramagnetic metals include the elements having atomicnumbers of 22 to 29, 42, 44 and 58-70. Examples of such metals arechromium (III), manganese (II), iron (II), iron (III), cobalt (II),nickel (II), copper (II), praseodymium (III), neodymium (III), samarium(III), gadolinium (III), terbium, (III), dysprosium (II), holmium (III),erbium (III) and ytterbium (III). Chromium (III), manganese (II), iron(III) and gadolinium (III) are preferred.

In one embodiment of the invention, two conjugates of ascorbic acid withDOTA were prepared, conjugates 22a and 22b. These conjugates have thefollowing structure:

These two conjugates were synthesized as described below.

DOTA-G-tri-t-butyl ester 4, an intermediate compound in the synthesis ofthe conjugate 22, was synthesized starting with glycine benzylesterhydrochloride (Aldrich), as described in Scheme 1. Chloroacetylchloride(Aldrich) was added to glycine benzylester hydrochloride in the presenceof K₂CO₃ to produce N-(chloroacetyl)-glycine benzyl ester 1. The ester 1was added to a suspension of DO3A-tri-t-butyl ester hydrochloride 2 (seeU.S. Pat. No. 5,573,752) in K₂CO₃ to produce DOTA-G-tri-t-butyl-benzylester 3. Subsequent catalytic hydrogenation produced theDOTA-G-tri-t-butyl ester 4.

Methyl 6-amino-6-deoxy-2,3-O-isopropylidene-2-keto-L-gulonate 16, whichwas a key protected intermediate for the synthesis of the conjugate 22,was synthesized starting from (2S,8S,1R,6R)-4,4,11,11-tetramethyl-3,5,7,10,12-pentaoxatricyclo[6.4.0.0<2,6>]dodecane-6-carboxylicacid 9. The compound was synthesized as described in Scheme 2. Thecommercially available acid 9 was methylated by MeI in the presence ofK₂CO₃. Mono-deprotection of the methyl ester 10 by Cu(OAc)₂ in H₂Oproduced the diol 11. The sulfite 12 was prepared from the diol 11 usingthionyl chloride in the presence of Et₃N. Oxidation of the sulfite 12with NaIO₄/RuCl₃ gave the cyclic sulfate 13. Treatment of the sulfate 13with NaN₃ in CH₃CN (acetonitrile), in the presence of Me₄N⁺Cl⁻ as aphase transfer catalyst, effected the ring opening with N₃ ⁻substitution to provide the azide 14. Subsequent hydrolysis andcatalytic hydrogenation of the azide 14 produced the desired amine 16 inan overall yield of 6.9%.

In an alternative embodiment for synthesizing compound 16, displacementof the —OTs group by N₃ ⁻ in 6-O-p-toluenesulfonyl-4-hydroxy-gulonate 17was achieved and is illustrated in Scheme 3. The mono-tosylate 17 wasprepared by treatment of the diol 11 with one equivalent of TsCl inpyridine in 63% yield. Tosylate 17 was treated with NaN₃ in DMF at 100°C. for 16 hours that produced the substituted azide 15 as a cleanproduct by TLC. The pure material 15 was isolated by silica columnchromatography providing a 52% yield. Subsequent catalytic hydrogenationproduced the desired amine 16 in an overall yield of 16.8%

The method of synthesis of the ascorbic conjugates from methyl (5S,7S,1R,6R)-7-(aminomethyl)-6-hydroxy-3,3-dimethyl-2,4,8-trioxabicyclo[3.3.0]octanecarboxlate16 is shown in Scheme 4. To obtain the desired conjugate 22a, the amine16 was first coupled with DOTA-G-tri-t-butyl ester 4 (synthesized as inScheme 1) in the presence of HATU/Et₃N to obtain compound 19a. Basichydrolysis of compound 19a produced the tris-t-Bu-DOTA-G-CH₂-gulonicacid conjugate 20a. Further deprotection of compound 20a wasinvestigated under several conditions as follows: 1) 6N HCl/THF 1/1,v/v, 45° C. for 7 h; 2) 4.5N H₂SO₄/THF (1/1, v/v), RT for 16 h; 3)TFA/H₂O (7/1, v/v), RT for 16 h. Of these methods, method 1 gave thehighest yield of 30% for the conjugate 21a, after purification by HPLC.Chelation with gadolinium produced the ascorbic-Gd chelate conjugate 22ain an overall yield of 13% from compound 16.

Following a similar approach, but by starting from6-[trans-4-(aminomethyl)-cyclohexyl-1-carbony]-amino-6-deoxy-2,3-isopropylidene-2-keto-gulonate18, the ascorbic-Gd chelate conjugate 22b was obtained, containing alonger linker residue in an overall yield of 4.8%. The final productsand intermediates were characterized by mass spectra, elementalanalysis, and NMR, as detailed in the Example section below. The6-[trans-4-(aminomethyl)-cyclohexyl-1-carbony]-amino-6-deoxy-2,3-isopropylidene-2-keto-gulonate18 was synthesized as shown in Scheme 5.

Relaxivity of a paramagnetic material in the presence of a large proteinsuch as human serum albumin maybe used to study the ability of thecompounds of the present invention to bind the target protein. As such,when a small molecule binds a large protein, the relaxivity of theformer will increase because of an increase in its rotationalcorrelation time. This increase in relaxivity may be used not only tomeasure the extent of binding but also to evaluate the viability of theparamagnetic agent as a blood pool contrast medium.

The relaxivity of the conjugates 22a and 22b in water as well as inwater containing a known amount (20%, v/v) of a HSA preparation, knownas, seronom (Table 1) were studied.

The relaxation time of the samples (22a or 22b) in seronom(T1_(Gd-ligand in seronom)) and in water (T1_(Gd-ligand in water)) weremeasured at 38° C. using an IBM PC/20 multispec relaxometer. Therelaxivity of the samples in seronom (r1_(Gd-ligand in seronom)) and inwater (r1_(Gd-ligand in water)) were calculated by the followingequations:r1_(Gd-ligand in seronom)=(1/T1_(Gd-ligand in seronom)−1/T1_(seronom))/[Gd-ligand]r1_(Gd-ligand in water)=(1/T1_(Gd-ligand in water)−1/T1_(water))/[Gd-ligand],

-   -   where [Gd-ligand] is the concentration of the chelate 22a or        22b, which was determined by ICP¹¹; T1_(seronon) is the        relaxation time for pure aqueous seronom; T1_(water) is the        relaxation time for pure water.

After the measurement, the sample in aqueous seronom was placed in acentrifree micropartition device (Millipore, Beverly, Mass.). The devicewas centrifuged at 500×g for 45 min in a fixed angle rotor (BeckmanModel J2-21M, JA-20 rotor). The solution (0.5 mL) was taken from belowthe filter (unbound Gd-ligand) for [Gd] ICP measurement. The [Gd] of theuncentrifuged sample was also measured as a control. The Fraction Boundwas calculated by the following equation:

FractionBound=([Gd-ligand]_(control)−[Gd-ligand]_(unbound))/[Gd-ligand]_(control).Table 1 details the results of the tests. TABLE 1 Relaxivity in waterRelaxivity in aqueous (mM⁻¹s⁻¹) seronom (mM⁻¹s⁻¹) Fraction Compoundr1_(Gd-ligand in water) r1_(Gd-ligand in seronom) bound 22a 4.83 6.388.8% 22b 7.20 8.18 10.9%

Table 1 shows that the increase in relaxivity in the presence of seronomis 32% and 14% in the case of 22a and 22b, respectively. Based on theseresults, it is believed that the binding of these conjugates with HSAmay not be strong enough for commercial blood pool MRI applications.However, the described conjugates 22a and 22b may be useful asextravascular MRI contrast agents. Moreover, the chemistry described andthe conjugates made herein may be used in other applications such asthose discussed herein and may be particularly useful where antioxidantproperties of ascorbic acid may be required.

It is understood that, for radiopharmaceutical or radiotherapyapplications, it is convenient to prepare the complexes of the presentinvention at, or near, the site where they are to be used. A single, ormulti-vial kit that contains all of the components needed to prepare thecomplexes of this invention, other than the radionuclide ion itself, isan integral part of this invention. The amount administered may beselected based on the desired use, such as to produce a diagnostic imageof an organ or other site of a subject or a desired radiotherapeuticeffect, by methods known in the art. Exemplary dosages are thoseemploying about 2-200 mCi rhenium, lutetium, or yttrium (forradiotherapy), or about 10-60 mCi technetium (for imaging).

Kits of the present invention comprise one or more vials containing thesterile formulation of a predetermined amount of a complexing ligand, anoxidant and optionally other components such as reducing agents,transfer ligands, buffers, lyophilization aids or bulking agents,stabilization aids, solubilization aids and bacteriostats. The inclusionof one or more optional components in the formulation will frequentlyimprove the ease of synthesis of the radiopharmaceutical by thepracticing end user, the ease of manufacturing the kit, the shelf-lifeof the kit, or the stability and shelf-life of the radiopharmaceutical.The improvement achieved by the inclusion of an optional component inthe formulation must be weighed against the added complexity of theformulation and added cost to manufacture the kit. The one or more vialsthat contain all or part of the formulation can independently be in theform of a sterile solution or a lyophilized solid.

Buffers useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of the radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids or bulking agents useful in the preparation ofdiagnostic kits useful for the preparation of radiopharmaceuticals areknown in the art and include lactose, sodium chloride, maltose, sucrose,PEG 8000, cyclodextrins, such as hydroxypropyl-γ-cyclodextrin (HP-γ-CD),dextran, Ficoll, and polyvinylpyrrolidine (PVP).

Stabilization aids, such as antioxidants, useful in the preparation ofradiopharmaceuticals and in diagnostic kits for the preparation ofradiopharmaceuticals include but are not limited to ascorbic acid,para-aminobenzoic acid (PABA), cysteine, monothioglycerol, sodiumbisulfite, sodium metabisulfite, gentisic acid, and inositol. Oneskilled in the art will appreciate that while conjugates of ascorbicacid are exemplified, the invention includes conjugates of otherantioxidants. Also, in addition to the covalent attachment of anantioxidant to a complexing ligand discussed herein, one or moreadditional stabilzation aids may be added to formulations of theconjugates of the invention.

Solubilization aids useful in the preparation of radiopharmaceuticalsand in diagnostic kits useful for the preparation of theradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monooloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. Preferred solubilizing aids are polyethyleneglycol, and Pluronics.

Bacteriostats useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of the radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or co-ligand and so forth.

The predetermined amounts of each component in the formulation aredetermined by a variety of considerations familiar to those skilled inthe art. These considerations are in some cases specific for thatcomponent and in other cases dependent on the amount of anothercomponent or the presence and amount of an optional component. Ingeneral, the minimal amount of each component is used that will give thedesired effect of the formulation. The desired effect of the formulationis that the practicing end user can synthesize the radiopharmaceuticaland have a high degree of certainty that the radiopharmaceutical can besafely injected into a patient and will provide diagnostic informationabout the disease state of that patient.

The present invention will be illustrated in greater detail by thefollowing specific examples. It is understood that these examples aregiven by way of illustration and are not meant to limit the disclosureor claims. Moreover, these examples are meant to further demonstratethat the synthesis of conjugates of ascorbic acid with macrocyclicpolyaminopolycarboxylates chelates. All percentages in the examples orelsewhere in the specification are by weight unless otherwise specified.

EXAMPLE 1

The synthesis of methyl(3aS,3bR,7aS,8aR)-2,2,5,5-tetramethyltetrahydro-8aH-[1,3]dioxolo[4,5]furo[3,2-d][1,3]dioxine-8a-carboxylate10 is detailed.

To a solution of(3aS,3bR,7aS,8aR)-2,2,5,5-tetramethyltetrahydro-8aH-[1,3]dioxolo[4,5]furo[3,2-d][1,3]dioxine-8a-carboxylicacid monohydrate 9 (10 g, 34.2 mmol, Aldrich) in DMF (anhydrous, 32 mL,Aldrich) was mixed with K₂CO₃ (3.4 g, 24.8 mmol, Aldrich). MeI (7.1 g,50 mmol, Aldrich) was added dropwise through a dropping funnel. Theresulting mixture was stirred at room temperature for 16 h. The solventwas removed in vacuo and the residue was dissolved in EtOAc (100 mL). Itwas washed with H₂O (2×60 mL), brine (1×50 mL) and dried over MgSO₄.EtOAc was evaporated. The crude material was purified by silica gelchromatography using EtOAc/Hexane to obtain product 10 (5.6 g; yield57%).

TLC: Silica gel, R_(f) 0.45, EtOAc/hexane 1/4. ¹HNMR (CDCl₃, ppm): 1.35,1.40, 1.50 (s, 12H, CH₃'s on the two isopropylidine rings); 3.85 (s, 3H,OCH₃); 4.08, (m, 2H, CH₂); 4.15, 4.30, 4.85 (s, 3H, CH's on C-3, C-4,C-5). Mass spectrum: 311.3 (M+Na)⁺; 289.3 (M+H)⁺.

EXAMPLE 2

The synthesis of Methyl(3aS,5R,6S,6aR)-6-hydroxy-5-(hydroxymethyl)-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate11 is detailed.

To a suspension of compound 10 (5.6 g, 18.3 mmol) in H₂O (65 mL) wasadded a solution of Cu(OAc)₂H₂O (25 mg, Aldrich) in H₂O (5 mL). It wasrefluxed (oil bath) for 15 min. The solution became clear. This solutionwas cooled and evaporated to dryness. Purification of the residue bysilica gel chromatography using EtOAc/Hexane afforded product 11 as awaxy solid (4.1 g; yield 90.3%).

TLC: Silica gel, R_(f) 0.70, EtOAc. ¹HNMR (CDCl₃, ppm): 1.40, 1.50 (s,611, CH₃'s on the isopropylidine ring); 3.85 (s, 3H, OCH₃); 3.98-4.00(m, 1H, CH on C-4); 4.10-4.13 (m, 1H, CH on C-5); 4.80 (s, 2H, CH₂);4.75 (m, 1H, CH on C-3). Mass spectrum: 271.2 (M+Na)⁺; 249.3 (M+H)⁺.

EXAMPLE 3

Two different synthesis processes of Methyl(3aR,5S,6R)-5-(azidomethyl)-6-hydroxy-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate15 are detailed.

EXAMPLE 3A From Sulfite 12 and Sulfate 13 (Scheme 2) i) Methyl(4aS,5aR,8aS,8bR)-7,7-dimethyltetrahydro-5aH-[1,3]dioxolo[4,5]furo[3,2-d][1,3,2]dioxathiine-5a-carboxylate2-oxide 12

To a solution of the diol 11 (3.4 g, 13.7 mmol) and triethylamine (0.58mol, 80 mL) in CH₂Cl₂ (40 mL) was added dropwise a solution of SOCl₂(2.3 g, 19.2 mmol, Aldrich) in CH₂Cl₂ (2 mL) at 0° C. It was stirred at0° C. for 15 min. It was then diluted with cold ether (80 mL), washedwith cold water (150 mL×2), dried over MgSO₄ and evaporated in vacuo.Purification by silica column chromatography afforded product 12 (1 g,yield 25%).

TLC: Silica gel, R_(f) 0.70, EtOAc/hexane 1/1. ¹HNMR (CDCl₃, ppm): 1.40,1.50 (s, 6H, CH₃'s on the isopropylidine ring); 3.90 (s, 3H, OCH₃); 4.2(d, 1H, CH on C-3); 4.31 (m, 1H, CH on C-5); 4.92 (m, 1H, CH on C-4);4.98 (m, 2H, CH on C-6).

Mass spectrum: 317.1 (M+Na)⁺.

ii) Methyl(4aS,5aR,8aS,8bR)-7,7-dimethyltetrahydro-5aH-[1,3]dioxolo[4,5]furo[3,2-d][1,3,2]dioxathiine-5a-carboxylate2,2-dioxide 13

To a solution of the sulfite 12 (1 g, 3.4 mmol) in CCl_(4/)CH₃CN (10 mL,1/1 v/v) was added a suspension of RuCl₃xH₂O (1 mg) and NaIO₄ (2.9 g,13.6 mmol) in H₂O (10 mL) at 0° C. The mixture was stirred vigorously atRT for 7 h. It was diluted with ether (20 mL), washed with water (30mL×2), sat. NaHCO₃ (30 mL×1), and dried over MgSO₄. Evaporation of thesolution afforded product 13 as a white crystalline solid (1 g, yield95%). The material was directly used without purification.

TLC: Silica gel, R_(f) 0.60, EtOAc/hexane 1/1. ¹HNMR (CDCl₃, ppm): 1.41,1.51 (s, 6H, CH₃'s on the isopropylidine ring); 3.88 (s, 3H, OCH₃); 4.38(s, 1H, CH on C-5); 4.85-4.98 (m, 2H, CH on C-6); 5.06 (s, 1H, CH onC-3); 5.20 (s, 1H, CH on C-4). Mass spectrum: 333.0 (M+Na)⁺.

iii) Conversion of sulfate 13 into azide 15

To a solution of the sulfate 13 (2 g, 6.45 mmol) in CH₃CN (10 mL) wasadded Me₄N⁺Cl⁻ (35 mg) and NaN₃ (2.1 g, 32.3 mmol). It was refluxed for4 h. The reaction was monitored for the disappearance of the startingsulfate by TLC. At the end of the reaction, the solvent was evaporated.To this residue was added dioxane (28 mL) and diluted H₂SO₄ (conc.H₂SO₄/H₂O 1/5 v/v, 2 mL). The mixture was stirred at RT for 16 h.Solvents were evaporated. The residue was dissolved in EtOAc (80 mL),washed with water (80 mL×2), dried over MgSO₄ and solvent evaporated todryness to obtain 15 (1 g, yield 57%).

TLC: Silica gel, R_(f) 0.70, EtOAc/hexane 3/7. ¹HNMR (CDCl₃, ppm): 1.40,1.50 (s, 6H, CH₃'s on the isopropylidine ring); 3.20 (d, 1H, OH); 3.50(q, 2H, CH₂); 3.75 (s, 3H, OCH₃); 4.18 (d, 1H, CH on C-4); 4.30 (m, 1H,CH on C-5); 4.65 (s, 1H, CH on C-3). Mass spectrum: 296.1 (M+Na)⁺.

EXAMPLE 3B From the tosylate 17 (Scheme 3) i) Methyl(3aS,5R,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4-methylphenyl)sulfonyl]oxy}methyl)dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate_(—)17

To a solution of 11 (1.0 g, 4.0 mmol) in pyridine (13 mL, Aldrich) at 0°C. (ice water bath) was added TsCl (0.76 g, 4.0 mmol, Aldrich) inportions. The resulting mixture was stirred at room temperature for 16h. The solvent was removed in vacuo and the residue was partitionedbetween EtOAc/H₂O. The EtOAc layer was washed with H₂O (2×40 mL), brine(1×40 mL) and dried over MgSO₄. EtOAc was evaporated and the crudematerial was purified by silica gel chromatography using EtOAc/Hexane.Product 17 was obtained as a white solid (1.0 g; yield 63%).

TLC: Silica gel, R_(f) 0.85, EtOAc/hexane 7/3. ¹HNMR (CDCl₃, ppm): 1.40,1.50 (s, 6H, CH₃'s on the isopropylidine ring); 2.44 (CH₃ on phenylring); 2.88 (d, 1H, OH); 3.85 (s, 3H, OCH₃); 4.19, 4.38 (m, 2H, CH₂);4.27 (d, 1H, CH on C-4); 4.50 (m, 1H, CH on C-5); 4.69 (s, 1H, CH onC-3); 7.35, 7.80 (d, 4H, CH's on phenyl ring). Mass spectrum: 425.2(M+Na)⁺; 403.2 (M+H)⁺.

ii) Conversion of tosylate 17 into azide 15

To a solution of 17 (400 mg, 1.0 mmol) in DMF (2 mL) was added NaN₃ (97mg, 1.5 mmol). The reaction mixture was heated at 100° C. (oil bath) for20 h. The solvent was removed in vacuo and the residue was partitionedbetween EtOAc/H₂O. The EtOAc layer was washed with H₂O (2×20 mL), brine(1×20 mL) and dried over MgSO₄. EtOAc was evaporated and the crudematerial was purified by silica gel chromatography using EtOAc/Hexane.Product 15 was obtained as a white solid (140 mg; yield 52%).

TLC: Silica gel, R_(f) 0.70, EtOAc/hexane 3/7. ¹HNMR (CDCl₃, ppm): 1.40,1.50 (s, 6H, CH₃'s on the isopropylidine ring); 3.20 (d, 1H, OH); 3.50(q, 2H, CH₂); 3.75 (s, 3H, OCH₃); 4.18 (d, 1H, CH on C-4); 4.30 (m, 1H,CH on C-5); 4.65 (s, 1H, CH on C-3). ¹³CNMR (CDCl₃, ppm): 25.0, 26.0(CH₃'s on the isopropylidine ring); 49.0 (CH₂); 53.5 (OCH₃); 74.0, 81.3,88.0 (CH's); 109.5, 114.5 (C on C-1 & C on the isopropylidine ring);168.2 (CO).

Mass spectrum: 296.1 (M+Na)⁺. Elemental Analysis: Found: C 44.34, H5.42, N 15.43%. Calculated for C₁₀H₁₅N₃O₆: C 43.96, H 5.53, N 15.38, O35.13%.

EXAMPLE 4

The synthesis of Methyl(3aR,5S,6R)-5-(aminomethyl)-6-hydroxy-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate16 is detailed.

To a solution of 15 (1.0 g, 3.7 mmol) in MeOH (30 mL) were added aqueousHCl (conc., 0.32 mL) and palladium, 10 wt. % (dry basis) on activatedcarbon (wet, Degussa type E101 NE/W, 307 mg, Aldrich). It washydrogenated at 22 psi for 0.5 h. The mixture was filtered through aCelite cake and the solvent evaporated to dryness. Product 16 wasobtained as an off white solid (1.0 g; yield 91.0%).

¹HNMR (CDCl₃, ppm): 1.40, 1.50 (s, 6H, CH₃'s on the isopropylidinering); 3.48-3.55 (m, 2H, CH₂); 3.80 (s, 3H, OCH₃); 4.48 (m, 1H, CH onC-4); 4.65 (m, 1H, CH on C-5); 4.80 (m, 1H, CH on C-3). ¹³CNMR (CDCl₃,ppm): 25.5, 27.0 (CH₃'s on the isopropylidine ring); 38.0 (CH₂); 53.5(OCH₃); 74.0, 77.5, 88.0 (CH's); 109.5, 114.5 (C on C-1 & C on theisopropylidine ring); 167.0 (CO).

Mass spectrum: 248.2 (M+Na)⁺.

EXAMPLE 5

The synthesis of methyl(3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate19a is detailed.

i) Synthesis of DOTA-G-tri-t-butyl ester (4) Preparation ofN-(chloroacetyl)-glycine benzyl ester (1)

To a suspension of glycine benzylester hydrochloride (25.2 g, 12.5 mmol,Aldrich) in CH₂Cl₂ (200 ml) was added a solution of K₂CO₃ (77 g, 55.8mmol) in H₂O (200 ml). The mixture was cooled to 0° C. andchloroacetylchloride (21.0 g, 18.6 mol, Aldrich) was added dropwise in15 min. The mixture was warmed to room temperature and stirred for 2 h.The two layers were separated and the aqueous layer was extracted withCH₂Cl₂ (100 ml). The organic layers were combined and washed with H₂O(100 ml), brine (100 ml), dried (MgSO₄). Evaporation of solvent affordeda light yellowish glassy solid. This was triturated with 250 ml ofhexane. The colorless solid was collected and dried to give 30.0 g ofthe material 1.

Yield: 100% TLC: R_(f) 0.8 (silica gel, 30% EtOAc/hexane). ¹HNMR(CDCl₃): δ 4.09 and 4.05 (2s, 6H, NCH₂ and COCH₂Cl); 5.18 (s, 2H,benzylic CH₂); 7.15 (s, 1H, NH); 7.34 (s, 5H, ArH).

Preparation of DOTA-G-tri-t-butyl-benzyl ester (3)

To a suspension of DO3A tri-t-butyl ester hydrochloride 2 (55.0 g, 100mmol) in acetonitrile (250 ml) was added anhydrous K₂CO₃(50.0 g, 360mmol). After 15 min. of stirring at room temperature a solution of 1(29.0 g, 120 mmol) in 50 ml of acetonitrile was added in 15 min. Themixture was stirred at room temperature for 24 h. K₂CO₃ was filtered offand solvent evaporated. The residue was dissolved in EtOAc (300 ml) andwashed with H₂O (150 ml), brine (100 ml) and dried (MgSO₄). The EtOAcsolution was concentrated to 150 ml in vacuo and 50 ml of hexane wasadded. The product crystallized out on keeping the solution at roomtemperature for 2 h. The crystals were filtered and dried. 51.5 g of thematerial 3 was obtained as a colorless crystalline solid.

Yield: 73%. TLC: R_(f) 0.55 (silica gel, 5% MeOH/CHCl₃). ¹HNMR(CDCl3): δ1.41(s,27 H, CH₃); 1.70-4.42 (26 H, CH₂); 5.10 (s, 2H, benzylic CH₂);7.34 (m, 5H, ArH); 9.53 (s, 1H, NH). Mass Spectrum: 720.4 (M+H)⁺, 742.3(M+Na)⁺, 664, 608, 552

Preparation of DOTA-G-tri-t-butyl ester (4)

To a solution of 3 (10.8 g, 15 mol) in MeOH (50 ml) was added palladium,10 wt. % (dry basis) on activated carbon (wet, Degussa type E101 NE/W,2.0 g). The mixture was hydrogenated at 50 psi for 4 h. The catalyst wasfiltered through a celite cake and evaporation of solvent afforded 8.8 gof 4 as a colorless solid.

Yield: 94% TLC: R_(f) 0.2 (silica gel, 5% MeOH/CHCl₃). HPLC system:Retention Time 18.63 min; Assay: >100% (area %); Column: YMC, C18;0.46×25 cm; solvent: Water(0.1% TFA)-Acetonitrile(0.1%TFA), Initialcondition, 15% ACN, Linear gradient to 55% ACN in 20 min and then to 90%CH₃CN in 40 min; Flow rate: 1.0 mL/min; Detection UV λ=220.¹HNMR(CDCl₃): δ 4.07(s, 27 H, CH₃); 1.80-4.35 (bm, 28 H, CH₂), 8.35 (s,1H, NH). Mass Spectrum: 630.4 (M+H)⁺, 652.4 (M+Na)⁺, 574, 518, 462.

ii) Synthesis of methyl(3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate19a

To a solution of 16 (0.9 g, 3.2 mmol) in CH₂Cl₂ (6 mL) was added 4 (2.0g, 3.2 mmol), HATU[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (1.2 g, 3.2 mmol, PerSeptive Biosystems), andtriethylamine (0.64 g, 6.4 mmol, Aldrich). The clear solution wasstirred at room temperature for 4 h. Solvents were evaporated and it wasdissolved in EtOAc (50 mL). It was washed with 5% NaHCO₃ (2×30 mL), 0.05N HCl (2×30 mL), H₂O (1×30 mL), and dried (MgSO₄). Evaporation ofsolvent afforded product 19a (2.3 g; yield 84%).

TLC: Silica gel, R_(f) 0.70, MeOH(CHCl₃ 1/4. ¹ (CDCl₃, ppm): 1.38-1.50(m, 33H, CH₃'s on t-Bu's & on isopropylidene ring); 1.90-3.60 (m, 28H,NCH₂COOtBu, NCH₂CH₂, CH₂ CONHCH₂CONH, CH₂CONHCH₂ CONH & CONHCH₂CONHCH₂); 3.70 (s, 3H, OCH3); 4.02, 4.25, 4.80 (m, 3H, CH's on the gulonicring); 6.75, 6.95 (t, 2H, NH's). Mass spectrum: 859.6 (M+H)⁺.

EXAMPLE 6

The synthesis of(3aR,5S,6R,6aS)-6-hydroxy-2,2-dimethyl-5-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylicacid 20a is detailed.

To a solution of 19a (1.0 g, 1.17 mmol) in MeOH (2 mL) was added asolution of LiOH.H₂O (48.5 mg, 1.17 mmol, Aldrich). The resultingsolution was stirred at room temperature for 4 h. Solvents wereevaporated and the residue was purified by silica column chromatography.Product 20a was obtained as a white solid (0.8 g; yield 81%).

TLC: Silica gel, R_(f) 0.25, MeOH/CHCl₃ 1/9. ¹HNMR (CDCl₃, ppm):1.38-1.50 (m, 33H, CH₃'s on t-Bu's & on isopropylidene ring); 1.90-3.60(m, 28H, NCH₂COOtBu, NCH₂CH₂, CH₂ CONHCH₂CONH, CH₂CONHCH₂ CONH &CONHCH₂CONHCH₂ ); 4.02, 4.25, 4.80 (m, 3H, CH's on the gulonic ring.Mass spectrum: 845.5 (M+H)⁺.

EXAMPLE 7

The synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-[2-[[2-[[(2R)-2-[(2S)-2,5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2-hydroxyethyl]amino]-2-oxoethyl]amino]2-oxoethyl]-21ais detailed.

20a (1.3 g, 1.58 mmol) was dissolved in 6N HCl/THF (1/1, v/v, 30 mL).The solution was stirred at 45° C. for 6.5 h. The solvents wereevaporated to dryness. The residue was dissolved in H₂O (30 mL) andpurified by preparative HPLC employing a YMC C-18 column. The column waseluted at 15 mL/min., 0% CH₃CN/H₂O (both containing 0.1% TFA). Fractionswere lyophilized to give pure 21a as a white fluffy solid (0.3 g; yield30.2%). ¹HNMR (D₂O, ppm): 2.90-4.05 (m, NCH₂COOH, NCH₂CH₂, CH₂CONHCH₂CONH, CH₂CONHCH₂ CONH, CONHCH₂CONHCH₂ , CHOH & OCH on ascorbicring). Mass spectrum: 619.3 (M+H)⁺. HPLC: Column: YMC C-18. Conditions:3% CH₃CN/H₂O (both containing 0.1% TFA), UW at 254 nm; flow rate 1.0mL/min.; t_(R): 5.14 min. Elemental Analysis: Found: C 36.91, H 4.56, N9.36%. Calculated for C₂₄H₃₈N₆O₁₃.2.4TFA.2H₂O: C 37.26, H 4.83, N 9.06,O 34.13, F 14.74%.

EXAMPLE 8

The synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-[2-[[2-[[(2R)-2-[(2S)-2,5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2-hydroxyethyl]amino]-2-oxoethyl]amino]2-oxoethyl]-,gadolinium salt 22a is detailed.

To a suspension of 21a (86 mg, 0.093 mmol) in H₂O (30 mL) was added 1NNaOH (Aldrich) solution to adjust the pH to 5. A solution of Gd(OAc)₃(62.2 mg, 0.15 mmol, Aldrich) in H₂O (5 mL) was added and the pH of themixture was maintained at pH 6 by adding 1N NaOH. The cloudy solutionwas stirred at room temperature for 16 h, then was warmed to 50° C. for4 h. The suspension was filtered and purified by preparative HPLCemploying a YMC C-18 column. The column was eluted at 15 mL/min., 0%CH₃CN/H₂O. Fractions were lyophilized to give pure material 22a as awhite fluffy solid (50 mg; yield 63.3%).

Mass spectrum: 774.2 (M+H)⁺. HPLC: Column: YMC C-18. Conditions: 3%CH₃CN/H₂O (both containing 0.1% TFA), UV at 254 nm; flow rate 1.0mL/min.; t_(R): 7.86 mm. Elemental Analysis: Found: C 33.81, H 4.87, N9.47, Gd 18.50%. Calculated for C₂₄H₃₅N₆O₁₃Gd4.5H₂O: C 33.76, H 5.19, N9.84, Gd 18.42, O 32.79%.

EXAMPLE 9

The synthesis of methyl(3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]amino}methyl)dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate 19b is detailed.

i) Synthesis of6-[trans-4-(aminomethyl)-cyclohexyl-1-carbony]-amino-6-deoxy-2,3-isopropylidene-2-keto-gulonate18 Preparation of4-{[(phenylmethoxy)carbonylamino]methyl}cyclohexanecarboxylic acid 23

To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (10 g,63.6 mmol, Aldrich) in 2N aqueous NaOH (65 ml) at 0° C. was added benzylchloroformate (11.9 g, 70 mmol, Aldrich) and the reaction temperaturewas maintained below 10° C. The cloudy mixture was stirred at RT for 0.5h. It was then diluted with H₂O (100 ml). The clear solution was washedwith ether (3×80 ml). The pH of the aqueous layer was adjusted to 2 byadding 6N HCl. The precipitates were filtered and dried in vacuo. 17.8 gof 23 was obtained as a white solid.

Yield: 96%. ¹HNMR (CDCl₃, ppm): 0.90-2.30 (m, 9H, CH₂'s & CH's oncyclohexyl); 2.20-2.30 (m, 1H, CHCOOH); 2.95-3.05 (m, 2H, NCH₂);4.80-4.85 (m, 1H, NH); 5.10 (s, 2H, PhCH₂ ); 7.30-7.40 (m, 5H, CH's onphenyl ring). Mass spectrum: (M+H)⁺ at 292.2.

Preparation ofmethyl(1S,5S,7S,6R)-6-hydroxy-3,3-dimethyl-2,4,8-trioxa-7-{[(4-{[(phenylmethoxy)carbonylamino]methyl}cyclohexyl)carbonylamino]methyl}bicyclo[3.3.0]octanecarboxylate 24

To a solution of 16 (2.0 g, 7.1 mmol) in CH₂Cl₂ (40 ml) was added 23(2.1 g, 7.1 mmol), HATU[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate](2.7 g, 7.1 mmol, PerSeptive Biosystems). It wascooled to 0° C. by ice-water bath. To this mixture triethylamine (1.43g, 14.2 mmol, Aldrich) was added and the mixture was stirred at 0° C.for 4 h. Solvents were evaporated and it was dissolved in EtOAc (100ml). It was washed with 5% NaHCO₃ (2×50 ml), 0.05 N HCl (2×50 ml), H₂O(1×50 ml), and dried (MgSO₄). Evaporation of the solvents and silica gelchromatography purification using MeOH/CHCl₃ afforded 3 g of 24 as awhite solid.

Yield: 81% TLC: Silica gel, R_(f) 0.50, MeOH/CHCl₃ 1/20. ¹HNMR (CDCl₃,ppm): 1.36, 1.46 (s, 6H, CH₃'s on isopropylidene ring); 0.85-0.95 (m,10H, CH₂'s and CH's on cyclohexyl); 2.98-3.02 (m, 2H, OCONHCH₂ ); 3.80(s, 3H, OCH₃); 3.95-4.15 (m, 2H, CONHCH₂ ); 4.52-4.85 (m, 3H, CH's onthe gulonic ring); 5.03 (s, 2H, CH₂Ph); 7.21-7.35 (m, 5H, CH's on phenylring). Mass spectrum: (M+H)⁺ at 521.3.

Preparation of6-[trans-4-(aminomethyl)-cyclohexyl-1-carbonyl-amino-6-deoxy-2,3-isopropylidene-2-keto-gulonate18

To a solution of 24 (2.9 g, 5.6 mmol) in MeOH (60 mL) was addedpalladium, 10 wt. % (dry basis) on activated carbon (wet, Degussa typeE101 NE/W, 1.0 g, Aldrich). The mixture was hydrogenated at 50 psi for16 h. Pd/C was filtered through a celite cake and solvent evaporated.2.0 g of the material 18 was obtained.

Yield: 93%. ¹HNMR (MeOH, ppm): 1.36, 1.46 (s, 6H, CH₃'s onisopropylidene ring); 0.95-2.15 (m, 10H, CH₂'s and CH's on cyclohexyl);2.65 (m, 2H, NH₂ CH₂ ); 3.20-3.30 (m, 2H, NCH₂); 3.60, 4.0, 4.25 (m,3H,CH's on the gulonic ring); 3.75 (s, 3H, OCH₃). Mass spectrum: (M+H)⁺ at387.2.

ii) Synthesis of methyl(3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]amino}methyl)dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylate 19b

To a solution of 18 (2.0 g, 5.2 mmol) in CH₂Cl₂ (30 mL) was added 6 (3.3g, 5.2 mmol), HATU (2.0 g, 5.2 mmol, PerSeptive Biosystems). The mixturewas cooled to 0° C. by ice-water bath and triethylamine (0.53 g, 5.2mmol, Aldrich) was added. The clear solution was stirred at 0° C. for 4h. Solvents were evaporated and the residue was dissolved in EtOAc (100mL). It was washed with 5% NaHCO₃ (2×50 mL), 0.05 N HCl (2×50 mL), H₂O(1×50 mL), and dried (MgSO₄). Evaporation of solvent and purification bysilica gel chromatography using MeOH/CHCl₃ afforded product 19b (1.5 g;yield 29%).

TLC: Silica gel, R_(f) 0.75, MeOH/CHCl₃ 1/10. ¹HNMR (CDCl₃, ppm):1.0-2.13 (m, 10H, CH₂'s & CH's on cyclohexyl ring); 1.43 (m, 27 H, CH₃'son t-Bu's); 1.40, 1.50 (s, 6H, CH₃'s on isopropylidene ring); 1.90-3.60(m, 28H, NCH₂COOtBu, NCH₂ CH₂, NCH₂ CH₂ , CH₂ CONHCH₂CONH, CH₂CONHCH₂CONH & CH₂ adjacent to cyclohexyl ring); 3.82 (s, 3H, OCH₃); 3.95 (m,2H, CH₂ adjacent to gulonic ring); 4.19, 4.90, 5.12 (m, 3H, CH's on thegulonic ring); 6.32, 6.48, 6.80 (t, 3H, NH's). Mass spectrum: 998.6(M+H)⁺.

EXAMPLE 10

The synthesis of(3aS,5S,6S,6aR)-6-hydroxy-2,2-dimethyl-5-({[(4-{[(N-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}glycyl)amino]methyl}cyclohexyl)carbonyl]amino}methyl)dihydrofuro[2,3-d][1,3]dioxole-3a(5H)-carboxylic acid 20b is detailed.

To a solution of 19b (1.5 g, 1.5 mmol) in MeOH (4 mL) was added asolution of LiOH.H₂O (63 mg, 1.5 mmol, Aldrich). The resulting solutionwas stirred at room temperature for 4 h. 30 mg of LiOH.H₂O was added andit was stirred at RT for 20 more hours. Solvents were evaporated toobtain crude product 20b (1.45 g; crude yield 99%). The material wasused without further purification.

TLC: Silica gel, R_(f) 0.20, MeOH/CHCl₃ 15/100. ¹HNMR (MeOH, ppm):0.85-2.05 (m, 43H, CH₂'s & CH's on cyclohexyl ring, CH₃'s on t-Bu's & onisopropylidene ring); 1.90-3.60 (m, 28H, NCH₂COOtBu, NCH₂ CH₂, NCH₂ CH₂, CH₂ CONHCH₂CONH,

CH₂CONHCH₂ CONH & CH₂ adjacent to cyclohexyl ring); 3.48 (m, 2H, NCH₂adjacent to gulonic ring); 3.90, 4.19, 4.40 (m, 3H, CH's on the gulonicring). Mass spectrum: 984.6 (M+H)⁺.

EXAMPLE 11

The synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-[2-[[2-[[[4-[[[(2R)-2-[(2S)-2,5-dihydro-3,4-dihydroxy-5-oxo-2-hydroxyethyl]amino]carbonyl]cyclohexyl]methyl]amino]-2-oxoethyl]amino]2-oxoethyl]-21bis detailed.

20b (1.41 g, 1.43 mmol) was dissolved in 6N HCl/THF (1/1, v/v, 30 mL).The solution was stirred at 45° C. for 6.5 h. The solvents wereevaporated to dryness. The residue was dissolved in H₂O (30 mL) andpurified by preparative HPLC employing a YMC C-18 column (250×30 mm).The column was eluted at 25 mL/min., 0% CH₃CN/H₂O (both containing 0.1%TFA) for 15 min., then 0%-20% in 60 min. Fractions were lyophilized togive pure 21b as a white fluffy solid (325 mg; yield 30%).

¹HNMR (D₂O, ppm): 0.78-2.20 (m, 10H, CH₂'s & CH's on cyclohexyl ring);2.90-3.95 (m, 32 H, NCH₂COOH, NCH₂ CH₂, NCH₂ CH₂ , CH₂ CONHCH₂CONH,CH₂CONHCH₂ CONH, CONHCH₂CONHCH₂ , CONHCH₂ CHOH, CHOH & OCH on ascorbicring). ¹³CNMR (D₂O, ppm): 31.9, 32.0. and 32.9 (four Cyclohexylmethylene carbons), 39.5 and 48.0 (two Cyclohexyl methine carbons),45.5, 46.3, and 48.0 (fifteen N—CH₂— carbons), 70.0 and 79.5 (twoascorbic ring methine carbons), 121.0 and 158.5 (two olefinic carbons),176.5, 183.5, and 184.6 (seven carbonyl carbons). Mass spectrum: 758.3(M+H)⁺. HPLC: Column: YMC C-18. Conditions: 7% CH₃CN/H₂O (bothcontaining 0.1% TFA), UV at 254 nm; flow rate 1.0 mL/min.; t_(R): 7.65min. Elemental Analysis: Found: C 42.81, H 5.77, N 9.82%. Calculated forC₃₂H₅₁N₇O₁₄.2.0TFA.H₂O: C 43.06, H 5.53, N 9.77%.

EXAMPLE 12

The synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-[2-[[2-[[[4-[[[(2R)-2-[(2S)-2,5-dihydro-3,4-dihydroxy-5-oxo-2-furanyl]-2-hydroxyethyl]amino]carbonyl]cyclohexyl]methyl]amino]-2-oxoethyl]amino]2-oxoethyl]-,gadolinium salt 22b is detailed.

To a suspension of 21b (100 mg, 0.132 mmol) in H₂0 (20 mL) was added 1NNaOH (Aldrich) solution to adjust the pH to 5. A solution of Gd(OAc)₃(58.99 mg, 0.15 mmol, Aldrich) in H₂O (5 mL) was added and the pH of themixture was maintained at pH 6 by adding 1N NaOH. The cloudy solutionwas stirred at room temperature for 16 h. The suspension was filteredand purified by preparative HPLC employing a YMC C-18 column (250×20mm). The column was eluted at 20 mL/min., 0% CH₃CN/H₂O, then 0%-20% in60 min. Fractions were lyophilized to give pure material 22b as a whitefluffy solid (68 mg; yield 56.4%).

Mass spectrum: 913.3 (M+H)⁺. ¹HPLC: Column: YMC C-18. Conditions: 7%CH₃CN/H₂O (both containing 0.1% TFA), UV at 254 nm; flow rate 1.0mL/min.; tR: 10.72 min. Elemental Analysis: Found: C 36.81, H 5.40, N9.11, Gd 15.09%; Calculated for C₃₂H₄₇N₇O₁₄GaNa.6.0H₂O: C 36.88, H 5.71,N 9.41, Gd 15.09%.

As previously stated, detailed embodiments of the present invention aredisclosed herein; however, it is to be understood that the disclosedembodiments are merely exemplary of the invention that may be embodiedin various forms. It will be appreciated that many modifications andother variations that will be appreciated by those skilled in the artare within the intended scope of this invention as claimed below withoutdeparting from the teachings, spirit and intended scope of theinvention.

1-127. (canceled)
 128. A method of stabilizing a radiopharmaceuticalligand, the method comprising covalently binding an antioxidant to aradiopharmaceutical ligand optionally including a targeting molecule.129. The method of claim 128, wherein the antioxidant is ascorbic acidor a derivative thereof.
 130. The method of claim 128, wherein theradiopharmaceutical ligand is Oxa-PnAO, N,N-dimethyl-Gly-Ser-Cys-Gly,N,N-dimethyl-Gly-t-butylGly-Cys-Gly, or DOTA.
 131. The method of claim128, wherein the radiopharmaceutical ligand is covalently bound to atargeting molecule.
 132. The method of claim 131, wherein the targetingmolecule is an amino acid, a peptide, a protein, or an antibody. 133.The method of claim 128, wherein the antioxidant is covalently bound toa targeting molecule.
 134. The method of claim 133, wherein thetargeting molecule is an amino acid, a peptide, a protein, or anantibody.
 135. The method of claim 128, wherein both the antioxidant andthe radiopharmaceutical ligand are covalently bound to a targetingmolecule.
 136. The method of claim 135, wherein the targeting moleculeis an amino acid, a peptide, a protein, or an antibody.
 137. The methodof claim 128, wherein the radiopharmaceutical ligand further comprises ametal selected from ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁶⁸Yb, ¹⁴⁰La,⁹⁰Y, ⁸⁸Y, ⁸⁶Y, ¹⁵³Sm, ¹⁶⁶HO, ¹⁶⁵Dy, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re,¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ²¹⁵Bi, 177Lu, chromium (III),manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper(II), praseodymium (III), neodymium (III), samarium (III), gadolinium(III), terbium, (III), dysprosium (III), holmium (III), erbium (III) andytterbium (111).
 138. The method of claim 137, wherein the metal is^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ⁸⁸Y, ⁸⁶Y, ¹⁷⁷Lu, or gadolinium (III). 139.The method of claim 128, wherein the antioxidant is

or a derivative thereof.
 140. The method of claim 128, wherein theantioxidant is

or a derivative thereof.
 141. The method of claim 128, wherein theantioxidant is 2-O-octadecylascorbic acid or a derivative thereof. 142.The method of claim 128, wherein the antioxidant is 6-hydroxy ascorbicacid or a derivative thereof.
 143. The method of claim 128, wherein theantioxidant is para-aminobenzoic acid (PABA) or a derivative thereof.144. The method of claim 128, wherein the antioxidant is cysteine or aderivative thereof.
 145. The method of claim 128, wherein theantioxidant is monothioglycerol or a derivative thereof.
 146. The methodof claim 128, wherein the antioxidant is gentisic acid or a derivativethereof.
 147. The method of claim 128, wherein the antioxidant isinositol or a derivative thereof.
 148. The method of claim 128, whereinthe antioxidant is sodium bisulfite.
 149. The method of claim 128,wherein the antioxidant is sodium metabisulfite.